The element nitrogen (N) is essential to all life forms on Earth and the availability of fixed nitrogen is often the limiting factor in food production. Biological nitrogen fixation, which occurs in a diverse group of microbes, represents the single largest input of fixed nitrogen in the reductive portion of the global nitrogen cycle. A molecular understanding of the enzyme responsible for biological nitrogen fixation, nitrogenase, could contribute to enhanced food production world wide, and thus to the overall health of the global population. Our goal is to contribute towards an understanding of how the two complex metallocenters of nitrogenase, the P cluster and FeMo-cofactor, are formed and function biologically. The project described herein will focus on the molecular mechanisms for P cluster and FeMo-cofactor formation, as well as the contribution of P clusters to the nitrogenase catalytic mechanism. These objectives will be accomplished through: (i) characterization of nif-specific gene products that function in the formation and insertion of FeMo-cofactor, (ii) characterization of the catalytic, spectroscopic, and kinetic properties of MoFe proteins that have been altered by site-directed mutagenesis or by the specific chemical modification of their P clusters. These experiments, which hetefore were either impossible or extremely difficult, can now be performed through the application of new purification procedures we have developed and on our ability to isolate an FeMo-cofactorless form of the MoFe protein that contains intact P clusters and can be activated to high levels of activity by FeMo-cofactor addition.